The fungus Candida albicans causes diverse infections with substantial morbidity and mortality. Invasive infec- tions cause 10,000 deaths per year in the US, and an estimated 400,000 deaths per year worldwide. The organism remains a threat for many reasons, including the limited scope of the antifungal armamentarium, the occurrence of drug resistance, the ability of the organism to grow as biofilm, and the diverse array of virulence determinants that enable it to infect almost any tissue. Our understanding of C. albicans infection mechanisms comes mainly from one clinical isolate, strain SC5314, and its derivatives. One chronic knowledge gap is the extent to which conclusions from analysis of SC5314 can be generalized to other clinical isolates. We have addressed this question in preliminary results through functional assays of genes that govern production of biofilm and hyphae in four additional clinical isolates, rep- resenting four major phylogenetic clades. Our findings indicate that the impact of simple deletions of well- studied genes is highly variable among C. albicans strains. We seek to extend our work to a larger panel of strains, to extend our focus to include host interaction phenotypes, and to use our findings to develop broadly applicable gene discovery strategies that exploit natural variation. In our first aim, we will address two key questions: First, is regulatory network architecture as diverse within C. albicans clades as it is between clades? Second, are species-wide common elements of a regulatory network enriched for functionally relevant target genes? We will extend our preliminary studies to include 15 additional clinical isolates that represent the major clades. We will use biological phenotypes to assess the uniformity of impact of the mutations, and gene ex- pression assays to make an appraisal of gene regulatory network variation. The utility of species-wide analysis will be tested in detail with the EFG1 gene; we will define strain-independent target genes and assess target gene function through assays of biofilm and hypha production. In our second aim, we will look at the biofilm- hyphal regulators from the perspective of virulence to determine whether and how the diverse impact of muta- tions extends to pathogenicity. We will analyze parameters of host interaction and infection trajectory via ex vivo and in vivo analyses. In our third aim, we will implement a new complementation-cloning strategy to iden- tify causal mutations for unique variant phenotypes from select clinical isolates. We will use clone library-based complementation in C. albicans itself. The overall results of these studies will provide a new view of key virulence-associated gene functions in C. albicans that spans the range of natural strain diversity. It will help to prioritize pathways and gene products as potential therapeutic targets due to their uniformly strong impact across C. albicans strains. The work will also provide two broadly applicable strategies, strain-independent network analysis and C. albicans-based com- plementation cloning, that yield new avenues to address multiple questions in the study of C. albicans virulence.